Name: implementing patch clamp and live fluorescence microscopy to monitor functional properties of freshly isolated pkd epithelium
Uploaded: 2015-09-01T12:00:00
Description: Watch this Scientific Journal Video about Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium at JoVE.com

作者想感谢格伦·斯洛克姆（威斯康星医学院）和科琳A.拉文（尼康仪器公司）与显微镜实验优秀的技术援助。这项研究是支持由美国国立卫生研究院授予R01 HL108880（以AS），R01 DK095029（以OPO）和K99 HL116603（以TSP），全国肾脏基金会IG1724（以TSP），美国心脏协会13GRNT16220002（以OPO）和本·J.里普斯研究奖学金由美国肾脏病学会（至DVI）。

下面描述的实验程序，在威斯康星州和德克萨斯大学健康科学中心休斯敦大学医学院获得批准的机构动物护理和使用委员会，并分别按照卫生指南实验动物的护理和使用国家研究院​​。 图1显示的组织分离和处理过程的主要步骤。简要地说，从PCK或SD大鼠的肾脏用于收集无论是从健康非拨小管或囊肿导管上皮单层的手动隔离。在这里，我们从4-16周龄PCK大鼠10,13研究肾脏。 ...

我们在这里描述的传统膜片钳技术和表面荧光钙成像的应用程序从ARPKD的鼠的遗传模型导出囊性上皮单层。该协议包括三个步骤，其中最应引起重视的囊肿（协议节的步骤1.5）的隔离和电生理研究。这些关键步骤需要大量的训练和耐心，读者不应该立刻感到沮丧。 首先，最要注意的囊肿单层隔离的过程。制备的这一部分需要手工技巧和显著影响进一步的工作，作为检体和其连...

离子通道在许多生理功能，包括细胞生长和分化中发挥显著作用。常染色体显性和隐性多囊肾疾病（ADPKD和ARPKD，分别）是其特征在于，所述的肾小管上皮细胞起源的肾流体填充囊肿的发展遗传性疾病。多囊肾是由PKD1或PKD2编码polycystins 1和参与细胞增殖和分化的调节如图2所示，膜蛋白的突变引起的。 PKD2本身或作为与PKD1一个复杂也发挥作为钙 -permeable阳离子通道1。所述PKHD1基因编码fibrocystin（涉及小管和/或维持上皮极性的纤毛相关受体样蛋白）的突变是ARPKD 2的遗传动力。囊肿生长是一个复杂的现象，伴随着不安扩散3,4，5血管生成，分化和极性损失性肾小管细胞6-8。 有缺陷的再吸收和分泌增强囊性上皮有助于在管腔和囊肿膨胀9,10-流体积聚。受损的流量依赖性钙离子浓度我的信号也已PKD 11-15时挂cystogenesis。 这里，我们描述适用于单通道活性和细胞内Ca 2+水平的囊性上皮单层从PCK大鼠分离的膜片钳测量的方法。这种方法成功地被我们用于表征上皮细胞钠离子通道（ENaC的）10的活性和钙离子浓度依赖我引起的钙 -permeable TRPV4和嘌呤信号级联13处理 。 在这些研究中，我们使用的PCK大鼠，模型ARPKD引起的在PKHD1基因的自发突变。该PCK应变originallÿ从的Sprague-Dawley（SD）大鼠16从而SD大鼠被用作适当的控制用于 ​​与PCK应变比较而得。其结果是，既大鼠肾段和非扩张集合管从同一PCK大鼠分离可以作为两个不同的比较组对囊性上皮实验。

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			<td style="width:219px;">TMC</td>
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			<td style="width:333px;">equipped with Faraday cage</td>
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implementing patch clamp and live fluorescence microscopy to monitor functional properties of freshly isolated pkd epithelium

期间多囊肾囊肿萌生和扩展是一个复杂的过程，其特征在肾小管细胞增殖，管腔流体积累和细胞外基质的形成异常。离子通道和细胞内钙信号的活动是决定的肾小管上皮细胞功能的关键生理参数。我们开发适合于实时观察离子通道的活性与在上皮单层从肾囊肿新鲜分离的细胞内Ca 2+水平膜片钳技术和登记的方法。 PCK大鼠的常染色体隐性遗传性多囊肾病（ARPKD）遗传模型，这里被用作离子通道和钙流的体外分析。这里描述的是详细的一步一步的步骤旨在隔离囊性单层和非扩张管从PCK或正常只SD（SD）大鼠，并监视单通道活性和细胞内Ca 2+动力学。此方法不需要酶加工，并允许在分析新鲜分离的上皮单层的天然环境。此外，这种技术是将细胞内钙的变化非常敏感，并生成高分辨率的图像进行精确的测量。最后，分离的囊性上皮可进一步用于与抗体或染料，制备原代培养物的纯化和关于各种生化分析染色。

潜在的ENaC参与cystogenesis已经证明了几项研究观察到了破坏表皮生长因子（EGF）在PKD进展22-25和异常钠重吸收在ARPKD鼠模型和组织培养26-28信令。例如，Veizis 等人表明，阿米洛利敏感的 Na +吸收在光盘细胞ARPKD 29的非直系同源BPK小鼠模型下降。我们最近证明，受损的钠和水的重吸收的孢囊是在加重cystogenesis 10的一个重要因素。具体来说，我们采用电生理...

Watch this Scientific Journal Video about Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium at JoVE.com

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

表现在肾小管上皮细胞离子通道在多囊肾病的病理一个显著的作用。这里，我们描述用于执行囊性上皮从啮齿动物肾脏新鲜分离的膜片钳分析和细胞内钙水平的测量的实验方案。

实施膜片钳和活荧光显微镜监视新鲜分离的PKD上皮细胞的功能特性

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal ...

Research

JoVE Journal

Biology

Methods for Patch Clamp Capacitance Recordings from the Calyx

膜片钳从花萼电容录音方法

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. ...

科研

生物学

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

补丁钳爪蟾卵母细胞中表达的离子通道

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Pressure-polishing Pipettes for Improved Patch-clamp Recording

改进的膜片钳记录的压力抛光移液器

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording.  We first developed this method for fabricating pipettes suitable for ...

Time-lapse Imaging of Mitosis After siRNA Transfection

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

基于荧光测定活细胞中的钙进入商店经营：从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

裂殖酵母的活细胞定量荧光显微镜分析

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

巨脂质体制剂成像和膜片钳电

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify ...

One-channel Cell-attached Patch-clamp Recording

单通道细胞贴附膜片钳记录

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

跨膜蛋白的重组，电压门控离子通道，KvAP，进入巨人单层囊的显微镜和膜片钳研究

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

单通道分析和钙成像在新鲜分离的肾小球足细胞

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells ...